Differential gene regulation underlies variation in melanic plumage coloration in the dark-eyed junco (Junco hyemalis).

Colour plays a prominent role in species recognition; therefore, understanding the proximate basis of pigmentation can provide insight into reproductive isolation and speciation. Colour differences between taxa may be the result of regulatory differences or be caused by mutations in coding regions of the expressed genes. To investigate these two alternatives, we studied the pigment composition and the genetic basis of coloration in two divergent dark-eyed junco (Junco hyemalis) subspecies, the slate-coloured and Oregon juncos, which have evolved marked differences in plumage coloration since the Last Glacial Maximum. We used HPLC and light microscopy to investigate pigment composition and deposition in feathers from four body areas. We then used RNA-seq to compare the relative roles of differential gene expression in developing feathers and sequence divergence in transcribed loci under common-garden conditions. Junco feathers differed in eumelanin and pheomelanin content and distribution. Within subspecies, in lighter feathers melanin synthesis genes were downregulated (including PMEL, TYR, TYRP1, OCA2 and MLANA), and ASIP was upregulated. Feathers from different body regions also showed differential expression of HOX and WNT genes. Feathers from the same body regions that differed in colour between the two subspecies showed differential expression of ASIP and three other genes (MFSD12, KCNJ13 and HAND2) associated with pigmentation in other taxa. Sequence variation in the expressed genes was not related to colour differences. Our findings support the hypothesis that differential regulation of a few genes can account for marked differences in coloration, a mechanism that may facilitate the rapid phenotypic diversification of juncos.

The effect of chronic and acute stressors, and their interaction, on testes function: an experimental test during testicular recrudescence.

Organisms are expected to invest less in reproduction in response to a stressor, but theory predicts that this effect should depend on the frequency and duration of stressors in the environment. Here, we investigated how an acute stressor affected testes function in a songbird, and how chronic stressors influenced the acute stress response. We exposed male dark-eyed juncos (Junco hyemalis) either to chronic or minimal (control) disturbance during testicular recrudescence, after which we measured baseline testosterone, testosterone after an acute handling stressor, and capacity to produce testosterone after hormonal stimulation. In a 2×2 design, we then killed males from the two chronic treatment groups either immediately or after an acute stressor to investigate the effect of long- and short-term stressors on the testicular transcriptome. We found that chronically disturbed birds had marginally lower baseline testosterone. The acute stressor suppressed testosterone in control birds, but not in the chronic disturbance group. The ability to elevate testosterone did not differ between the chronic treatments. Surprisingly, chronic disturbance had a weak effect on the testicular transcriptome, and did not affect the transcriptomic response to the acute stressor. The acute stressor, on the other hand, upregulated the cellular stress response and affected expression of genes associated with hormonal stress response. Overall, we show that testicular function is sensitive to acute stressors but surprisingly robust to long-term stressors, and that chronic disturbance attenuates the decrease in testosterone in response to an acute stressor.

Differential gene expression in seasonal sympatry: mechanisms involved in diverging life histories.

In an era of climate change, understanding the genetic and physiological mechanisms underlying flexibility in phenology and life history has gained greater importance. These mechanisms can be elucidated by comparing closely related populations that differ in key behavioural and physiological traits such as migration and timing of reproduction. We compared gene expression in two recently diverged dark-eyed Junco ( Junco hyemalis) subspecies that live in seasonal sympatry during winter and early spring, but that differ in behaviour and physiology, despite exposure to identical environmental cues. We identified 547 genes differentially expressed in blood and pectoral muscle. Genes involved in lipid transport and metabolism were highly expressed in migrant juncos, while genes involved in reproductive processes were highly expressed in resident breeders. Seasonal differences in gene expression in closely related populations residing in the same environment provide significant insights into mechanisms underlying variation in phenology and life history, and have potential implications for the role of seasonal timing differences in gene flow and reproductive isolation.

Potential for sexual conflict assessed via testosterone-mediated transcriptional changes in liver and muscle of a songbird.

Males and females can be highly dimorphic in metabolism and physiology despite sharing nearly identical genomes, and both sexes respond phenotypically to elevated testosterone, a steroid hormone that alters gene expression. Only recently has it become possible to learn how a hormone such as testosterone affects global gene expression in non-model systems, and whether it affects the same genes in males and females. To investigate the transcriptional mechanisms by which testosterone exerts its metabolic and physiological effects on the periphery, we compared gene expression by sex and in response to experimentally elevated testosterone in a well-studied bird species, the dark-eyed junco (Junco hyemalis). We identified 291 genes in the liver and 658 in the pectoralis muscle that were differentially expressed between males and females. In addition, we identified 1727 genes that were differentially expressed between testosterone-treated and control individuals in at least one tissue and sex. Testosterone treatment altered the expression of only 128 genes in both males and females in the same tissue, and 847 genes were affected significantly differently by testosterone treatment in the two sexes. These substantial differences in transcriptional response to testosterone suggest that males and females may employ different pathways when responding to elevated testosterone, despite the fact that many phenotypic effects of experimentally elevated testosterone are similar in both sexes. In contrast, of the 121 genes that were affected by testosterone treatment in both sexes, 78% were regulated in the same direction (e.g. either higher or lower in testosterone-treated than control individuals) in both males and females. Thus, it appears that testosterone acts through both unique and shared transcriptional pathways in males and females, suggesting multiple mechanisms by which sexual conflict can be mediated.

Highly context-specific activation of the HPG axis in the dark-eyed junco and implications for the challenge hypothesis.

One of the best studied hormone-behavior interactions is the transient rise in testosterone (T) associated with male-male aggression. However, recent research on songbirds has demonstrated numerous exceptions to this pattern.One species previously thought to elevate T in response to a simulated territorial intrusion is the dark-eyed junco (Junco hyemalis). Here, we show that under most circumstances male juncos do not elevate circulating T or CORT levels in response to social stimuli, despite being physiologically capable of elevating T as indicated by their response to GnRH. The lack of hormonal response was found regardless of the sex of the social stimulus (singing male vs. soliciting female), its sensory modality (song only, song + live lure, song + taxidermic mount), or the timecourse of sampling. Notably, males did elevate T levels when exposed to a simulated territorial intrusion in the days following simulated predation of their chicks. Whether the high T seen in these narrow circumstances represents stage-dependent social modulation of T or re-activation of male reproductive physiology in preparation for re-nesting (i.e. socially independent T modulation) remains to be determined. It is clear, however, that activation of the HPG axis is highly context-specific for male juncos. These results highlight important and unresolved issues regarding the socially mediated component of the challenge hypothesis and how it relates to the evolution of hormone-mediated traits.

De novo transcriptome sequencing in a songbird, the dark-eyed junco (Junco hyemalis): genomic tools for an ecological model system.

BACKGROUND: Though genomic-level data are becoming widely available, many of the metazoan species sequenced are laboratory systems whose natural history is not well documented. In contrast, the wide array of species with very well-characterized natural history have, until recently, lacked genomics tools. It is now possible to address significant evolutionary genomics questions by applying high-throughput sequencing to discover the majority of genes for ecologically tractable species, and by subsequently developing microarray platforms from which to investigate gene regulatory networks that function in natural systems. We used GS-FLX Titanium Sequencing (Roche/454-Sequencing) of two normalized libraries of pooled RNA samples to characterize a transcriptome of the dark-eyed junco (Junco hyemalis), a North American sparrow that is a classically studied species in the fields of photoperiodism, speciation, and hormone-mediated behavior. RESULTS: From a broad pool of RNA sampled from tissues throughout the body of a male and a female junco, we sequenced a total of 434 million nucleotides from 1.17 million reads that were assembled de novo into 31,379 putative transcripts representing 22,765 gene sets covering 35.8 million nucleotides with 12-fold average depth of coverage. Annotation of roughly half of the putative genes was accomplished using sequence similarity, and expression was confirmed for the majority with a preliminary microarray analysis. Of 716 core bilaterian genes, 646 (90 %) were recovered within our characterized gene set. Gene Ontology, orthoDB orthology groups, and KEGG Pathway annotation provide further functional information about the sequences, and 25,781 potential SNPs were identified. CONCLUSIONS: The extensive sequence information returned by this effort adds to the growing store of genomic data on diverse species. The extent of coverage and annotation achieved and confirmation of expression, show that transcriptome sequencing provides useful information for ecological model systems that have historically lacked genomic tools. The junco-specific microarray developed here is allowing investigations of gene expression responses to environmental and hormonal manipulations - extending the historic work on natural history and hormone-mediated phenotypes in this system.

A Vaccine to Prevent Egg Layer Peritonitis in Chickens.

A series of studies was undertaken in specific-pathogen-free white leghorn chickens for the development of a chicken model of avian pathogenic Escherichia coli (APEC) peritonitis. Once established, this model was then used to measure the effectiveness of a siderophore receptor and porin proteins (SRP®) APEC vaccine. Initially, five pilot studies were performed to compare the E. coli serotype, challenge route, and dose of inoculum that resulted in pathologies characteristic of the peritonitis observed in commercial layer facilities, such as widespread organ infection, atrophy, discoloration, corrugation of yolk sacs, and the presence of caseous exudate. Isolates of serotypes O1, O2, and O78 were tested by intravenous, intravaginal, intratracheal, and intraperitoneal routes and were compared at various levels of challenge inoculum. Daily observations of mortality and morbidity were made, and at necropsy, gross lesion scores were collected and bacterial colonization of internal organs determined. Outcomes varied from a complete lack of mortality or detectable pathology and low, or no, organ colonization in the case of intravaginal and intratracheal routes with each E. coli serotype to moderate to high levels of mortality, pathology, and colonization after challenge via the intravenous and intraperitoneal routes with O2 and O78 serotypes, respectively. The O78 serotype was found to result in pathologies consistent with field observations of peritonitis, and therefore, subsequent studies were performed only with O78. In addition to the relative failure with both the intratracheal and intravaginal routes of challenge, the intravenous route was found to be inconsistent and often resulted in lameness not observed with the intraperitoneal route. A final pilot study confirmed that the dose (∼ 8 log 10 CFU) administered by the intraperitoneal route replicated peritonitis, and therefore, all vaccination/challenge studies were conducted in this manner. Five vaccination/challenge studies are reported here in which variables of chicken age, vaccination interval, and vaccination to challenge interval were examined. In all studies, vaccine effectiveness was dramatic and was shown to completely protect against mortality and substantially against tissue colonization and pathology typical of APEC infections. The vaccine elicited a rapid onset of immunity with both narrow and broad vaccination intervals and in both young and mature chickens. Additionally, the vaccine was demonstrated to sustain robust effectiveness against mortality over 3 months. The SRP APEC vaccine should provide effective protection of young and mature chickens from E. coli under broadly flexible conditions of use in commercial operations.

Artíclo regular­Vacuna para prevenir la peritonitis de gallina de postura. Se llevó a cabo una serie de estudios en aves tipo Leghorn blancas libres de patógenos específicos para el desarrollo de un modelo en pollo para la peritonitis causada por Escherichia coli patógena aviar (APEC). Una vez establecido, este modelo se utilizó para medir la eficacia de una vacuna con proteínas del receptor de sideróforo y de porina (SRP®) de E. coli patógena aviar. Inicialmente, se realizaron cinco estudios piloto para comparar el serotipo de E. coli, la ruta de desafío y la dosis de inóculo que resultaron en patologías características de las peritonitis observadas en instalaciones comerciales de ponedoras, como la infección generalizada de órganos, atrofia, decoloración, ondulación de saco vitelino y la presencia de exudado caseoso. Los aislamientos de los serotipos O1, O2 y O78 se analizaron por vías intravenosa, intravaginal, intratraqueal e intraperitoneal y se compararon a varios niveles de inóculo de desafío. Se realizaron observaciones diarias de mortalidad y morbilidad, en la necropsia, se registraron puntuaciones de lesiones macroscópicas y se determinó la colonización bacteriana de los órganos internos. Los resultados variaron desde una ausencia total de mortalidad o patología detectable y una colonización de órganos baja o nula en el caso de las rutas intravaginal e intratraqueal con cada serotipo de E. coli hasta niveles de mortalidad, patología y colonización de moderados a altos después del desafío por vía intravenosa e intraperitoneal con los serotipos O2 y O78, respectivamente. Se encontró que el serotipo O78 dio como resultado patologías consistentes con las observaciones de campo de la peritonitis y por lo tanto, los estudios posteriores se realizaron solo con el serotipo O78. Además del fracaso relativo con las rutas de desafío intratraqueal e intravaginal, se descubrió que la vía intravenosa era inconsistente y a menudo, provocaba cojera que no se observaba con la vía intraperitoneal. Un estudio piloto final confirmó que la dosis (∼8 log10 UFC) administrada por vía intraperitoneal reproducía la peritonitis y por lo tanto, todos los estudios de vacunación/desafío se realizaron de esta manera. En este estudio se reportan cinco estudios de vacunación/desafío en los que se examinaron las variables de edad del pollo, intervalo de vacunación e intervalo de vacunación al desafío. En todos los estudios, la eficacia de la vacuna fue muy evidente y se demostró que protege completamente contra la mortalidad y sustancialmente contra la colonización de tejidos y la patología típica de las infecciones por E. coli patógena aviar. La vacuna provocó un rápido inicio de la inmunidad con intervalos de vacunación tanto estrechos como amplios y tanto en aves jóvenes como maduras. Además, se demostró que la vacuna mantiene una sólida eficacia contra la mortalidad durante tres meses. La vacuna con proteínas del receptor de sideróforo y de porina de E. coli patógena aviar debería proporcionar una protección eficaz de las aves jóvenes y maduras contra E. coli en condiciones de uso ampliamente flexibles en operaciones comerciales.

piRNA-mediated transposon regulation and the germ-line mutation rate in Drosophila melanogaster males.

Transposons, especially retrotransposons, are abundant in the genome of Drosophila melanogaster. These mobile elements are regulated by small RNAs that interact with the Piwi family of proteins-the piwi-interacting or piRNAs. The Piwi proteins are encoded by the genes argonaute3 (ago3), aubergine (aub), and piwi. Heterochromatin Protein 1 (HP1), a chromatin-organizing protein encoded by the Suppressor of variegation 205 [Su(var)205] gene, also plays a role in this regulation. To assess the mutational impact of weakening the system for transposon regulation, we measured the frequency of recessive X-linked lethal mutations occurring in the germ lines of males from stocks that were heterozygous for mutant alleles of the ago3, aub, piwi, or Su(var)205 genes. These mutant alleles are expected to deplete the wild-type proteins encoded by these genes by as much as 50%. The mutant alleles of piwi and Su(var)205 significantly increased the X-linked lethal mutation frequency, whereas the mutant alleles of ago3 did not. An increased mutation frequency was also observed in males from one of two mutant aub stocks, but this increase may not have been due to the aub mutant. The increased mutation frequency caused by depleting Piwi or HP1suggests that chromatin-organizing proteins play important roles in minimizing the germ-line mutation rate, possibly by stabilizing the structure of the heterochromatin in which many transposons are situated.

Behavioral effects of social challenges and genomic mechanisms of social priming: What's testosterone got to do with it?

Social challenges from rival conspecifics are common in the lives of animals, and changes in an animal's social environment can influence physiology and behavior in ways that appear to be adaptive in the face of continued social instability (i.e. social priming). Recently, it has become clear that testosterone, long thought to be the primary mediator of these effects, may not always change in response to social challenges, an observation that highlights gaps in our understanding of the proximate mechanisms by which animals respond to their social environment. Here, our goal is to address the degree to which testosterone mediates organismal responses to social cues. To this end, we review the behavioral and physiological consequences of social challenges, as well as their underlying hormonal and gene regulatory mechanisms. We also present a new case study from a wild songbird, the dark-eyed junco (Junco hyemalis), in which we find largely divergent genome-wide transcriptional changes induced by social challenges and testosterone, respectively, in muscle and liver tissue. Our review underscores the diversity of mechanisms that link the dynamic social environment with an organisms' genomic, hormonal, and behavioral state. This diversity among species, and even among tissues within an organism, reveals new insights into the pattern and process by which evolution may alter proximate mechanisms of social priming.

Assessing impacts of unconventional natural gas extraction on microbial communities in headwater stream ecosystems in Northwestern Pennsylvania.

Hydraulic fracturing and horizontal drilling have increased dramatically in Pennsylvania Marcellus shale formations, however the potential for major environmental impacts are still incompletely understood. High-throughput sequencing of the 16S rRNA gene was performed to characterize the microbial community structure of water, sediment, bryophyte, and biofilm samples from 26 headwater stream sites in northwestern Pennsylvania with different histories of fracking activity within Marcellus shale formations. Further, we describe the relationship between microbial community structure and environmental parameters measured. Approximately 3.2 million 16S rRNA gene sequences were retrieved from a total of 58 samples. Microbial community analyses showed significant reductions in species richness as well as evenness in sites with Marcellus shale activity. Beta diversity analyses revealed distinct microbial community structure between sites with and without Marcellus shale activity. For example, operational taxonomic units (OTUs) within the Acetobacteracea, Methylocystaceae, Acidobacteriaceae, and Phenylobacterium were greater than three log-fold more abundant in MSA+ sites as compared to MSA- sites. Further, several of these OTUs were strongly negatively correlated with pH and positively correlated with the number of wellpads in a watershed. It should be noted that many of the OTUs enriched in MSA+ sites are putative acidophilic and/or methanotrophic populations. This study revealed apparent shifts in the autochthonous microbial communities and highlighted potential members that could be responding to changing stream conditions as a result of nascent industrial activity in these aquatic ecosystems.

Testosterone affects neural gene expression differently in male and female juncos: a role for hormones in mediating sexual dimorphism and conflict.

Despite sharing much of their genomes, males and females are often highly dimorphic, reflecting at least in part the resolution of sexual conflict in response to sexually antagonistic selection. Sexual dimorphism arises owing to sex differences in gene expression, and steroid hormones are often invoked as a proximate cause of sexual dimorphism. Experimental elevation of androgens can modify behavior, physiology, and gene expression, but knowledge of the role of hormones remains incomplete, including how the sexes differ in gene expression in response to hormones. We addressed these questions in a bird species with a long history of behavioral endocrinological and ecological study, the dark-eyed junco (Junco hyemalis), using a custom microarray. Focusing on two brain regions involved in sexually dimorphic behavior and regulation of hormone secretion, we identified 651 genes that differed in expression by sex in medial amygdala and 611 in hypothalamus. Additionally, we treated individuals of each sex with testosterone implants and identified many genes that may be related to previously identified phenotypic effects of testosterone treatment. Some of these genes relate to previously identified effects of testosterone-treatment and suggest that the multiple effects of testosterone may be mediated by modifying the expression of a small number of genes. Notably, testosterone-treatment tended to alter expression of different genes in each sex: only 4 of the 527 genes identified as significant in one sex or the other were significantly differentially expressed in both sexes. Hormonally regulated gene expression is a key mechanism underlying sexual dimorphism, and our study identifies specific genes that may mediate some of these processes.

Variation in candidate genes CLOCK and ADCYAP1 does not consistently predict differences in migratory behavior in the songbird genus Junco.

Recent studies exploring the molecular genetic basis for migratory variation in animals have identified polymorphisms in two genes ( CLOCK and ADCYAP1) that are linked to circadian rhythms and correlate with migratory propensity and phenology among individuals and populations. Results from these initial studies are mixed, however, and additional data are needed to assess the generality and diversity of the molecular mechanisms that regulate the biology of migration. We sequenced CLOCK and ADCYAP1 in 15 populations across the two species of the avian genus Junco, a North American lineage in which multiple recently diverged subspecies and populations range from sedentary to long-distance migrants. We found no consistent associations between allele length and migratory status across the genus for either CLOCK or ADCYAP1. However, within two subspecies groups, populations that migrate longer distances have longer CLOCK alleles on average. Additionally, there was a positive relationship between ADCYAP1 allele length and migratory restlessness (zugunruhe) among individuals within one of two captive populations studied-a result similar to those reported previously within captive blackcaps ( Sylvia atricapilla). We conclude that, while both ADCYAP1 and CLOCK may correlate with migratory propensity within or among certain populations or species, previously identified relationships between migratory behavior and sequence variants cannot be easily generalized across taxa.

Maintenance of MHC Class IIB diversity in a recently established songbird population.

We examined variation at MHC Class IIB genes in a recently established population of dark-eyed juncos (Junco hyemalis) in a coastal urban environment in southern California, USA relative to an ancestral-range population from a nearby species-typical montane environment. The founding population is estimated to have been quite small, but we predicted that variation at the major histocompatibility complex (MHC) among the founders would nevertheless be preserved owing to the high functional significance of MHC. Previous studies of MHC in songbirds have had varying degrees of success in isolating loci, as passerines show extensive MHC gene duplication. In order to compare diversity in the two populations, we employed two published approaches to sequencing MHC Class II exon 2: direct sequencing with exon-based primers, and traditional cloning and sequencing with intron-based primers. Results from both methods show that the colonist population has maintained high levels of variation. Our results also indicate varying numbers of alleles across individuals, corroborating evidence for gene duplication in songbird MHC. While future studies in songbirds may need to take a genomic approach to fully understand the structure of MHC in this lineage, our results show that it is possible to use traditional methods to reveal functional variation across populations.